Immunomodulatory properties of bip

ABSTRACT

The present invention relates to substances having immunomodulatory properties and to the use of such substances for the treatment or prevention of an unwanted immune response. In particular, the present invention relates to the use of BiP or a functional fragment of homolog thereof, or a nucleic acid molecule encoding BiP or a functional fragment or homolog thereof, in the treatment or prevention of an unwanted immune response.

[0001] The present invention relates to substances havingimmunomodulatory properties and to the uses of such substances for thetreatment or prevention of an unwanted immune response. The presentinvention also relates to a pharmaceutical composition comprising asubstance having immunomodulatory properties.

[0002] There are many situations in which unwanted immune responses leaddirectly to disease or interfere with therapy. Autoimmune diseases areone class of diseases of the first kind. Examples of the second kind ofsituation are responses which interfere either with the function oftransplanted tissues and organs (allo- or xeno-graft rejection) or whichinactivate substances used for gene therapy and which have notpreviously been encountered by the immune system of the recipient. Asimilar example of the latter kind is the response following theinfusion of therapeutic proteins collectively known as ‘biologics’ ofhuman or non-human origin, including monoclonal antibodies and othertherapeutic proteins such as blood clotting factors, and enzymes. Thesevaried situations are at present poorly managed and each demandsseparate therapeutic approaches.

[0003] The therapeutic approach of the present invention is based onBiP, the 78 kD endoplasmic reticulum chaperone. In International patentapplication PCT/GB99/03316 (publication No. WO 00/21995), it has beendemonstrated that recombinant BiP, expressed and purified fromtransfected E. coli was able to prevent the induction ofcollagen-induced arthritis (CIA) in susceptible DBA/1 mice. Theisolation of BiP from human cells and cell lines has been fullydescribed in WO 00/21995 as well as the cloning and expression of theDNA encoding this protein. The skilled person is therefore referred toWO 00/21995 for all necessary information relevant to the presentapplication. WO 00/21955 is thus incorporated herein by reference. TheBiP protein from human cells has a high degree of homology with BiP fromother species and the term BiP is therefore used herein to embrace allsuch proteins which have the property of inducing IL-10. Minorvariations on the specific DNA and amino acid sequences disclosed in WO00/21995 are also to be included provided the above property is retainedas discussed further herein.

[0004] The inventors have researched the mode of action of the BiPprotein and have confirmed that it has a general immunomodulatoryproperty which is of application to the treatment of diseases other thanrheumatoid arthritis. In particular, the inventors have found that BiPcan be used in preventing adjuvant arthritis (AA) in Lewis rats. As canbe seen from the experiments described below, BiP was able tosignificantly inhibit the development of AA.

[0005] The present invention provides the use of BiP or a functionalfragment or homolog thereof, or a nucleic acid molecule encoding BiP ora functional fragment or homolog thereof, in the manufacture of amedicament for the treatment or prevention of an unwanted immuneresponse.

[0006] It is preferred that the use according to the present inventionis not for the treatment or prevention of rheumatoid arthritis (RA) orcollagen-induced arthritis (CIA).

[0007] It is further preferred that the use of the present invention isfor preventing an unwanted immune response.

[0008] The term “BiP” as used herein refers to the 78 kD endoplasmicreticulum chaperone protein as defined in WO 00/21995. It has been foundthat BiP causes: CD14+ cells to release IL-10; stimulates CD8+ cells toproliferate and release IL-10; inhibits the recall antigen response; andactivates the expression of an array of anti-inflammatory genes inmonocytes, including the migration inhibitory factor (MIF), the solubleTNF receptor II and TIMPs. Preferably, the BiP protein has the aminoacid sequence given in WO 00/21995 as SEQ ID NO. 1 or SEQ ID NO. 2.

[0009] The term “a functional fragment” as used herein refers to afragment of BiP which is capable of eliciting at least part of anactivity of the full BiP protein. In particular, it is preferred thatthe functional fragment has at least one of the following functions:causes CD14+ cells to release IL-10; stimulates CD8+ cells toproliferate and release IL-10; inhibits the recall antigen response; oractivates the expression of an array of anti-inflammatory genes inmonocytes, including the migration inhibitory factor (MIF), the solubleTNF receptor II and TIMPs. Preferably the functional fragment is atleast 20 amino acids, more preferably at least 50 amino acids and mostpreferably at least 100 amino acids in length. Particularly preferredfragments comprise a conserved region which has been found to behomologous to a number of naturally occurring BiP proteins. Suchconserved regions are considered to have a specific function.

[0010] The term “a functional homolog” as used herein refers to ahomolog that retains at least part of an activity of the BiP proteindescribed in WO 00/21995. In particular, it is preferred that thefunctional homolog has at least one of the following functions: causesCD14+ cells to release IL-10; stimulates CD8+ cells to proliferate andrelease IL-10; inhibits the recall antigen response; or activates theexpression of an array of anti-inflammatory genes in monocytes,including the migration inhibitory factor (MIF), the soluble TNFreceptor II and TIMPs. It is preferred that the functional homolog hasat least 80%, more preferably at least 90% and most preferably at least95% amino acid sequence homology with one of the BiP proteins describedin WO 00/21995. Preferably the sequence homology is measured by usingBLAST analysis. It is particularly preferred that the functional homologdiffers by only 1 to 20 amino acids from one of the BiP proteinsdescribed in WO 00/21995. It is further preferred that the amino acidchanges are conservative. Conservative changes are those that replaceone amino acid with one from the family of amino acids which are relatedin their side chains. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarconservative replacement of an amino acid with a structurally relatedamino acid will not have a major effect on the biological activity ofthe protein. Mutations which increase the number of amino acids whichare capable of forming disulphide bonds with other amino acids in theprotein are particularly preferred in order to increase the stability ofthe protein.

[0011] The nucleic acid molecule used in the present invention encodesBiP or a functional fragment or homolog thereof as defined above. Thenucleic acid molecule can be obtained by methods well known in the art.For example, naturally occurring sequences may be obtained by genomiccloning or cDNA cloning from suitable cell lines or from DNA or cDNAderived directly from the tissues of an organism, such as a human ormouse. Positive clones may be screened using appropriate probes for thenucleotide molecule desired. PCR cloning may also be used. The probesand primers can be easily generated given that the sequence of BiP isknown (see WO 00/21995). Preferably the nucleic acid molecule has thesequence given in WO 00/21995 as SEQ ID NO. 3.

[0012] Numerous standard techniques known in the field of molecularbiology may be used to prepare the desired nucleic acid or the probesand primers for identifying the positive clones. The nucleotidemolecules probes or primers may be synthesised completely using standardoligonucleotide synthesis methods, such as the phosphoramidite method.

[0013] Numerous techniques may be used to alter the nucleic acidsequence obtained by the synthesis or cloning procedures, and suchtechniques are well known to those skilled in the art. For example, sitedirected mutagenesis, oligonucleotide directed mutagenesis and PCRtechniques may be used to alter the DNA sequence. Such techniques arewell known to those skilled in the art and are described in the vastbody of literature known to those skilled in the art, for exampleSambrook et al., (1989).

[0014] The nucleic acid is preferably in the form of a vector comprisingthe necessary elements leading to the expression of the nucleic acidsequence encoding BiP or a functional fragment or homolog thereof. Forexample, it is preferred that the vector comprises a promoter operablylinked to the nucleic acid sequence and a transcription terminationsequence. Suitable promoters, transcription termination sequences andother functional elements required to obtain expression of the nucleicacid are well known to those skilled in the art.

[0015] The nucleic acid may be delivered to the individual using anymethod. For example, the nucleic acid may be delivered as a free nucleicacid, in the form of a viral delivery vector such as an adenovirus,contained in a liposome or via any known method.

[0016] The unwanted immune response may be any unwanted immune response.Specific unwanted immune responses are discussed in details below.

[0017] A Prevention of Unwanted Immune Responses

[0018] i) Prevention of Rejection of Allo- and Xeno-Transplants

[0019] The rejection of allo- and xeno-transplants (TX) is a majorproblem barrier in the more effective use of TX for the therapy of organfailure. Present anti-rejection regimens are expensive, requirelife-long administration, may produce toxic side effects and are notuniversally effective. TX are grafted at a precisely known time. Just asthe inventors have shown that BiP is able to prevent CIA and AA ifgiven, respectively, at the time of or before the induction ofarthritis, it is recommended to administer BiP just before or at thetime of TX to prevent rejection. TX that may be beneficially treated inthis way include all TX of tissues and organs, whether solid (forexample, liver, kidney) or single cell (for example, blood cells, bonemarrow cells or stem cells).

[0020] ii) Prevention of Immune Response to Biologic TherapeuticSubstances

[0021] A range of biologic therapies are used in clinical medicine.These include products from non-human sources and products from humansources. The biologics may be purified from a natural source, producedby recombinant gene technology, secreted after transfection of genes, orsynthesised. These biologics may be proteins, glycoproteins or complexsugars. A disadvantage is that this has the potential to induce animmune response when administered to an immunologically naïveindividual.

[0022] There are two main consequences of the induction of such animmune response. The first is the development of anaphylactic shock thatmay be life threatening. The second is the loss of therapeutic activityof the product because of the development of neutralising antibodiesduring the course of the immune response. It is recommended toadminister BiP before or at the time of these interventions in order toprevent the development of this unwanted immune response and thusprevent these therapeutic failures.

[0023] B Treatment of Existing Diseases

[0024] Existing immune-mediated diseases such as type I diabetesmellitus (in the early phase before complete destruction of the betacells in the islets of Langerhans), thyroiditis, multiple sclerosis anddiseases in which the immune system is activated are also open totreatment by the parenteral administration of BiP. This is because BiPreleases IL-10 and other regulatory molecules from target cells such asCD14+ monocytes and CD8+ T cells.

[0025] According to a first embodiment of the use of the presentinvention the unwanted immune response is associated with animmune-mediated disease. Immune-mediated diseases include auto-immunediseases. Specific immune-mediated diseases include type-1 diabetes,thyroididtis, multiple sclerosis, systemic lupus erythematosus, Crohn'sdisease and all forms of viral and autoimmune hepatitis.

[0026] It is preferred that the use according the first embodiment ofthe present invention additionally comprises the use of an agent forenhancing the treatment or prevention of the immune-mediated disease inthe manufacture of the medicament.

[0027] The agent for enhancing the treatment or prevention of theimmune-mediated disease may be any agent including IL-10, IL-4, IL-11,TGF-beta, IL-13 and soluble cytokine receptors such as IL-1Ra, IL-1 andTNF soluble receptors.

[0028] Preferably the medicament is for administration to an individualsuffering from or susceptible to developing an immune-mediated disease.Methods are know for determining whether an individual is suffering froman immune-mediated disease and methods are known for determining if anindividual is likely to develop an immune-mediated disease. Methods fordetermining whether an individual is likely to develop an immune includeanalysing risk factors such as genetic markers or environmentalinfluences such as diet, etc.

[0029] In use, the medicament obtained by the use according to the firstembodiment of the present invention is preferably administered to anindividual before the immune-mediated disease develops or as soon as theimmune-mediated disease has been diagnosed.

[0030] According to a second embodiment of the use of the presentinvention the unwanted immune response is associated with the rejectionof a transplanted organ, tissue or cells.

[0031] The rejection response is well know and occurs when donatedtissue is recognised as foreign by the recipient's immune system. Therejection response occurs with transplanted organs, such as heart, lung,kidney, liver, etc., transplanted tissues, such as skin, muscle tissue,etc., and with transplanted cells, such as bone marrow cells and stemcells.

[0032] It is preferred that the use according the second embodiment ofthe present invention additionally comprises the use of an agent forenhancing the treatment or prevention of the immune response associatedwith rejection of a transplanted organ, tissue or cells, in themanufacture of the medicament.

[0033] The agent for enhancing the treatment or prevention of the immuneresponse associated with the rejection of transplanted organs, tissue orcells may be any agent that suppresses the immune system includingglucorticoids, cyclosporin A, azathioprine, rapamycin and tacrolimus.

[0034] In use, the medicament obtained by the use according to thesecond embodiment of the present invention is preferably administered toan individual before or at substantially the same time as thetransplantation of the organ, tissue or cells.

[0035] According to a third embodiment of the use of the presentinvention the unwanted immune response is the immune response to abiologic.

[0036] A biologic is any therapeutic agent given to an individual. Thebiologic may be from non-human or human sources. The biolgic may be aprotein molecule (i.e. an enzyme, an antibody molecule, receptor ligand,etc), a glycoprotein, a polypeptide, peptide, carbohydrate, or anorganic or inorganic chemical compound.

[0037] The use of biologics can cause unwanted immune responses. Forexample, an immune response can be raised against the biologic which mayprevent the therapeutic activity of the biologic. Alternatively, theimmune response may be so large that it lead to anaphylatic shock. Forexample, anti-TNFα therapy has resulted in the shortening of theinterval between dosing (infliximab) this increasing the cost, and itsuse has been limited by anaphylaxis.

[0038] It is preferred that the use according the third embodiment ofthe present invention additionally comprises the use of an agent forenhancing the treatment or prevention of the immune response to thebiologic.

[0039] The agent for enhancing the treatment or prevention of the immuneresponse to the biologic may be any agent that suppresses the immunesystem including glucorticoids, cyclosporin A, azathioprine, rapamycinand tacrolimus.

[0040] In use, the medicament obtained by the use according to the thirdembodiment of the present invention is preferably administered to anindividual before or at substantially the same time as the biologic.

[0041] The present invention also provides the use of BiP or afunctional fragment or homolog thereof, or a nucleic acid moleculeencoding BiP or a functional fragment or homolog thereof, forstimulating the release of IL-10 from cells capable of releasing IL-10.Preferably the cells are peripheral blood mononuclear cells (PBMCs).Preferably, the PMBCs are CD14+ monocytes and/or CD8+ T cells and/orCD4+ T cells.

[0042] Preferably BiP or a functional fragment or homolog thereof, or anucleic acid molecule encoding BiP or a functional fragment or homologthereof, is used to stimulate the release of IL-10 from PBMCs in vitroor ex vivo.

[0043] Preferably BiP or a functional fragment or homolog thereof, or anucleic acid molecule encoding BiP or a functional fragment or homologthereof is used to additionally stimulates gene expression of at leastone of monocyte migration inhibitory factor (MIP), soluble TNF receptorII, IL-10 anti-inflammatory mediators and tissue inhibitor of matrixmetalloproteinases (TIMP). Preferably BiP or a functional fragment orhomolog thereof, or a nucleic acid molecule encoding BiP or a functionalfragment or homolog thereof, does not stimulate gene expression ofmatrix metalloproteinases (MMPs), monocyte chemoattractant protein(MCP-1) or TNFα.

[0044] The present invention also provides a pharmaceutical preparationcomprising BiP or a functional fragment or homolog thereof, or a nucleicacid molecule encoding BiP or a functional fragment or homolog thereof,in combination with a pharmaceutically acceptable carrier for use in thetreatment or prevention of an unwanted immune response.

[0045] Preferably the pharmaceutical composition additionally comprisesan agent for enhancing the treatment or prevention of the unwantedimmune response.

[0046] The pharmaceutical composition of the present invention comprisesa therapeutically effective amount of BiP or a functional fragment orhomolog thereof, or a nucleic acid molecule encoding BiP or a functionalfragment or homolog thereof. The term “therapeutically effective amount”as used herein refers to an amount of a therapeutic agent needed totreat or prevent the unwanted immune response.

[0047] For any agent, the therapeutically effective dose can beestimated initially either in cell culture assays, or in animal models,usually mice, rabbits, dogs, or pigs. The animal model may also be usedto determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

[0048] The precise effective amount for a human subject will depend uponthe severity of the disease state, general health of the subject, age,weight, and gender of the subject, diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. This amount can be determined by routineexperimentation and is within the judgement of the clinician. Generally,an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05mg/kg to 10 mg/kg.

[0049] Pharmaceutical compositions of this invention comprise BiP or afunctional fragment or homolog thereof, or a nucleic acid moleculeencoding BiP or a functional fragment or homolog thereof, with anypharmaceutically acceptable carrier, adjuvant or vehicle.Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene polyoxypropylene-block polymers,polyethylene glycol and wool fat.

[0050] The pharmaceutical compositions of this invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.Oral administration or administration by injection are preferred. Thepharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intrasynovial, intrastemal, intrathecal, intralesional and intracranialinjection or infusion techniques.

[0051] The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as Ph. Helv or a similar alcohol.

[0052] The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

[0053] The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a novel agent of thisinvention with a suitable non irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

[0054] Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the novel agents of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active agent suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-transdermal patches arealso included in this invention.

[0055] The pharmaceutical compositions of this invention may beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

[0056] The present invention also provides a method of treating orpreventing an unwanted immune response comprising administrating to anindividual in need of such treatment an effective dose of thepharmaceutical composition according to the present invention.

[0057] As indicated above, there are many routes of administration ofBiP or a functional fragment or homolog thereof, or a nucleic acidmolecule encoding BiP or a functional fragment or homolog thereof,including intravenous, intramuscular, nasal, oral, cutaneous, andtopical. In particular, details of several preferred approaches to usingBiP or a functional fragment or homolog thereof, or a nucleic acidmolecule encoding BiP or a functional fragment or homolog thereof aredescribed below.

[0058] (a) Induction of Mucosal Tolerance.

[0059] Delivery of BiP (p78) autoantigen or functional peptides derivedtherefrom by mucosal routes, e.g. through the intestine or nasal mucosa,alters the immune response by downregulating disease activity leavingthe patient's immune system otherwise intact. Alternatively p78 orfunctional p78 peptides can be delivered as a nucleic acid moleculeencoding them within an appropriate mammalian expression vector.

[0060] (b) Vaccination with TCR Peptides

[0061] Peptides of the CDR3 region of the T cell receptor Valpha andVbeta chains can be synthesised and used as vaccines for delivery byintradermal or intramuscular injection (see Kotzin et al., ArthritisRheum., 11, 1906-1919, 1998). BiP or a functional fragment or homologthereof can be used in the same way.

[0062] (c) MHC Blockade with Native or Altered Peptides

[0063] BiP or a functional fragment or homolog thereof, may be givenparenterally or orally in appropriate cases either unmodified ormodified by amino acid substitution and/or attachment of chemicalgroupings so as to block MHC and especially HLA-DR4 thereby leading tosuppression of T cell activation and disease. BiP or a functionalfragment or homolog thereof, may be combined with soluble HLA-DR4molecules and applied parenterally or orally.

[0064] (d) Induction of Tolerance by Plasmid DNA Immunisation

[0065] Plasmids consisting of nucleic acid coding for BiP or afunctional fragment or homolog thereof, may be given by injection. DNAcoding for human IL-10, IL-4, IL-11, or TGF-beta, incorporated singly orin any combination, may be used to deviate the immune response to BiPtowards a TH2 mode so as to suppress disease.

[0066] The present invention is now described by way of example onlywith reference to the following Figures:

[0067]FIG. 1 shows the prevention of adjuvant arthritis by BiP. MaleLewis rats (n=5) were immunised with 50 μg PBS/DDA in each hind footpad(ie 100 μg/rat). Control animals (n=5) received only the PBS/DDAmixture. Thirteen days later, adjuvant arthritis was induced by a singleintradermal injection of 0.5 mg M. tuberculosis in 100 μl IFA in thebase of the tail.

[0068]FIG. 2 shows the results of the investigation into BiP binding toperipheral blood mononuclear cell (PBMC) populations and fibroblast likesynoviocytes by double immunofluorescence. Column A shows PBMC stainedwith human serum albumin (HSA) fluorescein isothiocyanate (FITC), as thenegative control, and column B, FITC conjugated BiP, the PBMC weredouble stained with CD14, CD20, CD4, CD8, CD56. Column C shows BiP.FITCbinding to 2 rheumatoid arthritis fibroblast-like synoviocytes comparedwith the HSA.FITC negative control.

[0069]FIG. 3 shows IL-10 production following culture of peripheralblood mononuclear cells (PBMC) with BiP (20 μg/ml), beta-galactosidase(b-gal) (20 μg/ml) or lipopolysaccharide (LPS) (20 ng/ml) in the absence(A) or presence (B) of polymixin B for 24 hours. Culture supernatantswere collected and IL-10 was measured by ELISA.

[0070]FIG. 4 shows the proliferation of CD8 clone FC2B5 to BiP (closedcircles) and control antigen β-galactosidase (open circles). This clonewas generated from the peripheral blood of a normal individual. Thisprofile is representative of other BiP responsive clones.

[0071]FIG. 5 shows the cytokine profiles of BiP responsive clones andlines. The cytokine levels were measured in supernatants of cellspreviously shown to be BiP responsive, stimulated by mitogen. Theprofiles are compared with irradiated feeder cells alone (first dataset).

[0072]FIG. 6 shows the proliferative response of T cells to BiPstimulation.

[0073]FIG. 7 shows BiP-driven T cell cytokine production from animalsimmunised with BiP.

[0074]FIG. 8 shows BiP driven T cell cytokine production from controlanimals.

[0075]FIG. 9 shows the uptake of tritiated thymidine following anallogeneic reaction between peripheral blood monocytes (MO) eithercultured for 5 days in tissue culture medium, or matured withgranulocyte macrophage-colony stimulating factor (GM-CSF)+IL-4, or withGM-CSF+IL-4+BiP, prior to irradiation and culture with allogeneicperipheral blood mononuclear cells.

[0076]FIG. 10 shows the proliferation response of peripheral bloodmononuclear cells measured by the uptake of tritiated thymidine ineither unstimulated cultures (TCM) or cultures stimulated with BiP (20μg/ml) or tuberculin PPD (10 μg/ml) or with BiP and PPD.

EXAMPLES

[0077] Materials and Methods

[0078] Fluorescein Isothiocyanate Labelling of Proteins

[0079] BiP or human serum albumin (HSA) were prepared at a concentrationof 2 mg/ml in carbonate buffer 0.1M pH9.6. A stock solution offluorescein isothiocyanate (FITC) was prepared at 10 mg/ml in carbonatebuffer 0.1M pH 9.6. 50 μg FITC/mg protein was added to the proteinsolution in a glass container covered in foil. The solution was placedon a circular mixer and incubated at room temperature for 2 hours. TheFITC labelled protein was then placed in dialysis tubing (which had beenboiled for 5 mins with each of three changes of fresh distilled water)and dialysed overnight in 5 litres of phosphate buffered saline (PBS)(0.15M NaCl, 4 mM NaH₂PO₄, 0.01M Na₂HPO₄ pH 7.2) followed by two furtherchanges of 5 litres PBS. FITC labelled proteins were aliquoted andstored at 4° C.

[0080] Immunfluorescent Staining of Cells

[0081] PBMC were separated on Lymphoprep (Nycomed Amersham, Amersham,UK) by density centrifugation at 800 g and then washed three times inHanks buffered saline solution (Life Technologies, Paisley UK).Fibroblast-like synoviocytes or other adherent cells grown in culturewere gently scraped from the surface of the flask beforeimmunofluorescenct staining. The cells were pelleted and resuspended inPBS containing 0.5% bovine serum albumin and 0.01% azide (PBS/BSA/az).Cells at 10⁵-10⁶/ml were used for the staining. 100 μl of cells wereplaced in a tube and 10 μl of ⅕ normal human serum added. The cells areincubated on ice for 10 minutes and then washed twice at 300 g inPBS/BSA/az at 4° C. The required amount of the FITC conjugated proteinwas then added to the cells in conjunction with any other proteindirectly conjugated to a different fluorochrome, such as phycoerythrin(PE), and the tube vortexed. The actual amount of protein added must bedetermined for each conjugation by a dose response curve. The cells wereincubated on ice for 20 minutes and then washed twice at 300 g inPBS/BSA/az at 4° C.

[0082] BiP.FITC was used at {fraction (1/50)} dilution andcounter-stained with anti-CD20 (B cell marker), CD3, CD4, CD8 (T cellmarkers), CD56 (NK cell marker), or CD14 (monocyte marker) all directlyPE conjugated and used at 5 μl/100 μl (Becton Dickinson, Oxford, UK).After the final wash the cells were fixed in PBS/BSA/az with 1%paraformaldehyde in 250 μl aliquots. The cells were then analysed on aFACScan using Cellquest software (Becton Dickinson, Oxford, UK).

[0083] Determination of Cytokine Production by PBMC Stimulated with BiP

[0084] Supernatants from cultures PBMC (10⁶1/ml) either unstimulated orstimulated by BiP (20 μg/ml) were harvested after 24 hours incubation.All cytokines were measured by commercial ELISA obtained fromPharmingen, Oxford, UK following 24 h stimulation.

[0085] Cloning Procedure for BiP Responsive Cells

[0086] Cloning of Specific T Cells:

[0087] Mononuclear cells were plated at 1×10⁶ cells ml⁻¹ in 2 ml culturewells in the presence of 20 μgml⁻¹ BiP (this concentration has beenpreviously shown to be optimal for proliferation) in culture medium(RPMI 1640+10% human serum, L-glutamine and penicillin and streptomycin.Cells were cultured at 37° C. in 5% CO₂. After 7 days

[0088] Lymphocult-T (LC-T) was added to the cultures (40 μlml⁻¹) as asource of interleukin-2 (IL-2). After a further 7 days 1×10⁶ irradiatedautologous feeder cells were added to each well with 40 μlml⁻¹ LC-T and20 μgml⁻¹ BiP. This regime was continued for 3 rounds of feeder cellsand then the cells plated at 10 cells per well into 96U plates with1×10⁴ γ-irradiated allogeneic feeder cells (4000 Rads: ¹³⁷Cesium source)and 2 μgml⁻¹ Phytohaemagglutinin (PHA). After one week LC-T was added tothe wells (40 μlml⁻¹) and after a further week 1×10⁴ irradiated feedercells, Lymphocult-T and PHA was added again. The cells were expandedinto progressively bigger wells using this regime, until sufficient cellnumbers were achieved for further study.

[0089] Proliferation Assays.

[0090] When sufficient cell numbers were achieved 1×10⁴ cloned cellswere incubated for three days with 1×10⁵ irradiated autologous feedercells in the presence or absence of BiP (20 μgml⁻¹) or PHA (2 μgml⁻¹).

[0091] The cells were incubated for the last 18 hours with ³H-thymidine(0.2 μCi) and then harvested. Proliferation was expressed as astimulation index (SI): proliferation in the presence ofstimulant/proliferation in the presence of medium alone.

[0092] Determination of Clonality:

[0093] Phenotypic analysis was carried out on responding clones usingCD3, 4 and 8 (BD). Briefly, cells were washed in FACS buffer (phosphatebuffered saline containing 1% bovine serum albumin and 0.05% sodiumazide) and incubated with 4 μl of the appropriate antibodies.Three-colour analysis was performed using a FACScan flow cytometer andcell-quest software. T cell receptor usage was determined using a panelof both FITC conjugated and non-conjugated antibodies. Briefly, forconjugated antibodies 1×10⁴ cells were incubated with 4 μl of each ofthe FITC conjugated V beta specific antibodies. Clones were also stainedwith 4 μl of both anti-CD4 conjugated to FITC and CD8 conjugated to PE.Where non-conjugated antibodies were used cells were incubated for 40mins on ice with the primary antibody, washed twice in FACS buffer, thenincubated for a further 40 mins with a FITC conjugated goat antibodyraised against mouse immunoglobulins.

[0094] Stained cells were run on a FACScan flow cytometer with a 488 nmlaser and the results analysed using Cellquest and WinMDI analyticalsoftware.

[0095] Cytokine Determination.

[0096] Supernatants were removed from cultures 24 hours after the lastround of stimulation. Supernatants from cultures containing onlyirradiated feeders, LC-T and PHA were used as controls. The amount ofinterleukin (IL) 4 and IL-1, and γ-interferon and tumour necrosisfactor-α were determined by ELISA (Pharmingen, according tomanufacturers instructions). Briefly, plates (Nunc Maxisorp) were coatedwith a cytokine specific capture antibody, blocked with 10% foetal calfserum (FCS: to stop non-specific binding) and the supernatants incubatedon the plate overnight at 4° C. Bound cytokine was detected with abiotin conjugated detection antibody and visualised with streptavidinconjugated horseradish peroxidase and TMB.

[0097] Inhibition of Recall Antigen Responses by PBMC Preincubated withBiP

[0098] PBMC were cultured either alone or with BiP (20 μg/ml) for 0, 24,48 or 72 hours prior to the addition of tuberculin purified proteinderivative (PPD)(10 μg/ml) and the cells incubated for 6 days followingthe addition of PPD. Tritiated thymidine was added to the cultures forthe final 6 hours. The cells were then harvested and the uptake oftritiated thymidine assayed using a dry matrix beta counter(Canbarra-Packard, Pangbourne, UK).

[0099] Cytokine Expression Array:

[0100] Monocytes (MO) were separated by negative selection using animmunomagnetic bead kit (Dynal, Wirral, UK) and placed in culture at2.10⁶/ml for 24 hours either alone or stimulated with BiP(20 μg/ml) orwith PMA (10 ng/ml)+IONO(250 ng/ml). The supernatants were harvested forELISA and the cells were processed for extraction of total RNA andproduction of cDNA using oligo d(T) primers and reverse transcriptase.The expression array (R&D Systems, Oxford, UK) was used according to themanufacturer's instructions.

[0101] Mechanism of Action of BiP in Preventing AA and CIA

[0102] A Stimulating Release IL-10 from Human CD14+ Monocytes

[0103] The ability of BiP to prevent CIA and AA suggested to theinventors that it may have a generic ability to downregulate immuneresponses. In order to test this possibility, BiP was conjugated tofluorescein isothiyocyanate (FITC) and used in flow cytometry. As can beseen from FIG. 2, BiP binds to different populations of cells found inhuman peripheral blood but especially to human CD14+ monocytes.

[0104] When peripheral human blood mononuclear cells were cultured withBiP, cells were stimulated to release interleukin 10 (IL-10) as well astumour necrosis factor (TNF)α (see FIG. 3). It is important to note thatthe amounts of secreted IL-10 are significantly greater than those forTNFα. Since IL-10 is anti-inflammatory and downmodulates immuneresponses while TNFα is pro-inflammatory, the administration of BiP willlead to the induction of an anti-inflammatory and downmodulatory immuneenvironment thus explaining its ability to prevent the induction of CIAand AA.

[0105] B Stimulating Release from CD8+ T cells

[0106] CD8+ cells from peripheral blood of humans subjects may bestimulated by BiP to proliferate (FIG. 4). Indeed, the inventors havegenerated clones of such CD8+ T cells. These clones do not secrete thepro-inflammatory cytokine interferon (IFN)γ but do secrete IL-10 (FIG.5). Thus these cells have the characteristics of Tcl regulatory CD8+ Tcells and are able to downmodulate immune responses.

[0107] C Inhibition of Recall Antigen Responses by PBMC Pre-Treated withBiP

[0108] Pre-incubation of PBMC with BiP induced inhibition of theresponse to tuberculin purified protein derivative (PPD), a recallantigen. The inhibition increased with the length of time the cells hadbeen exposed to BiP prior to the PPD challenge. (Inhibition of PPDresponses: PBMC+BiP, Oh, 44.5±30%, range 0-67%; 24 h, 47.2±35.8%, range0-87%; 48 h, 59.2±27%, range 30-90%; 72 h, 64±23.6%, range 33-90%)

[0109] D BiP Induces Monocytes to Activate a More Anti-InflammatoryArray of Genes Compared with Activation by Phorbol Myristic Acid (PMA)and Calcium Ionophore (IONO).

[0110] An expression array (R&D Systems, Oxford, UK) capable ofscreening 375 different genes was used to analyse the gene activationprofile of resting monocytes or that following BiP or PMA+IONOactivation. The preliminary results show that those genes activated byBiP were more anti-inflammatory than those activated following directcell activation by PMA+IONO. Differences are shown in Table 1. To benoted should be the following facts: BiP specifically induced geneactivation for monocyte migration inhibitory factor (MIF), soluble TNFreceptor II and IL-10 anti-inflammatory mediators, upregulated tissueinhibitor of matrix metalloproteinases (TIMP) but did not induce matrixmetalloproteinase (MMP) or monocyte chemoattractant protein (MCP)-1,potent pathogenic mediators, or the inflammatory cytokine, TNFα. Incontrast, PMA+IONO activation of monocytes upregulated many inflammatorymediators, such as TNF α, MCP-1 and MMP-1, 9 and -10. TABLE 1 Rest MOBiP MO BiP MO PMA/IONO MO Chemokines and receptors ENA-78 Y** Y Y YMIP-1alpha Y* Y Y MIP-1beta Y* Y GRO alpha Y Y Y Y* GRO beta Y Y Y Y*GRO gamma Y Y Y Y* IL-8 Y* Y* Y* Y** MCP-1 Y MCP-3 Y CCR1 Y CXCR4 Y LDGFY Y Y PARC Y Y Orphan receptors RDC-1 Y Y ChemR23 Y Y Y PARC Y YProteinases or related factor Caspase Y Y MMP-1 Y MMP-9 Y MMP-10 YTIMP-1 Y Y Y Y Urokinase R Y Y* Y* Y Caspase 1 Y Y Integrins Integrinbeta 1 Y Y Y Y Integrin beta-2 Y Y Y Y Integrin beta-4 Y Y Y TGFsuperfamily Activin A Y Y Y Y* TNF superfamily LIGHT Y Y Y Y Cytokinesand inhibitors IL-1beta Y** Y** Y** Y** IL-1Ra Y Y Y Y IL-6 Y Y Y YIL-10 Y TNF alpha Y GM-CSF Y MIF Y Y Cytokine receptors IFNgamma RI YIFNgamma RII Y Y IL-7R alpha Y Y Y TNFRII Y CD14 Y* Y Y Y

[0111] Expression array of the genes activated in resting monocytes(REST MO), or stimulated by BiP(BiP MO), or by PMA+IONO (PMA+IONO MO). Yindicated the gene was activated and mRNA was present, * indicated ahigh level of expression. Only the positive genes have been included inthe table.

[0112] In Vitro Functional Studies with BiP

[0113] The functional consequences of the addition of BiP has beeninvestigated in two important experimental systems:

[0114] 1) The allogeneic reaction is the in vitro equivalent oftransplantation

[0115] 2) Tuberculin purified protein derivative (PPD) stimulation is ameasure of the lymphocytes response to recall antigens (an antigen towhich the subject has already been immunised).

[0116]FIG. 9 shows that allogeneic peripheral blood lymphocytes react tothe resting monocytes with a low response. When the monocytes arematured into dendritic cells, using the well established technique ofadding granulocyte macrophage colony stimulating factor (GM-CSF) andinterleukin-4 (IL-4) for 5 days, the response by the allogeneic PBMC isgreatly increased. When BiP is added with GM-CSF+IL-4 at the start ofmonocyte maturation the allogeneic response by PBMC is decreased to thatof the background control.

[0117] These findings support the concept that BiP given before organ,tissue, cell, gene or protein administration will prevent immunisation.In terms of transplantation this should lead to acceptance of the graft.

[0118]FIG. 10 shows that the PPD response by PBMC is significantlyreduced to background levels when BiP is added to the PPD cultures.

[0119] These findings support the concept that the administration of BIPwill suppress ongoing inflammatory and autoimmune diseases.

[0120] In Vivo Experiments

[0121] In developing BiP as a therapeutic drug two important propertiesneed to be ascertained: first, the nature of the cytokines releasedafter parenteral administration of BiP and, second, the optimum dose ofBiP needed to achieve the maximum release of cytokines

[0122] 1 BiP as an Immunomodulator

[0123] Method: Male DBA-1 mice (8-12 weeks old) were immunisedsubcutaneously (s.c.) with 200 μg of BiP in phosphate buffered saline(PBS). PBS alone or bovine serum albumin (BSA) were administered ascontrols. 14 days later, spleens and lymph nodes were removed and T cellcultures set up and stimulated with varying concentrations of BiP at0.1, 1, 10, 20 μg/ml. After 4 days of culture, the pro-inflammatorycytokine interferon (IFN)-γ and the anti-inflammatory cytokinesinterleukin (IL)4, IL-5 and IL-10 were assayed. T cell proliferation wasassessed by ³H-thymidine incorporation.

[0124] Results: T cells derived from animals immunised with BiPproliferated in a dose dependent manner on in vitro stimulation (FIG. 6,BiP). Control animals exhibited modest proliferation to in vitrostimulation with BiP (FIG. 6, PBS). The cytokine profiles alsodemonstrated a dose dependent increase in IL-4 and IL-5 production by Tcells derived from all BiP immunised mice (levels ˜500 pg/ml and ˜700pg/ml respectively) (FIG. 7) compared to the control animals (<100pg/ml) (FIG. 8). However, only modest levels of IFN-γ were produced by Tcells derived from BiP-only immunised mice.

[0125] Conclusion: BiP is surprisingly immunogenic and immunising micewith BiP results in a raised anti-inflammatory cytokine profile with aswitch towards a TH2 profile.

[0126] 2 Optimising the BiP Immunisation Dose

[0127] Methods: Groups of mice were immunised with 4 different doses ofBiP (either 50, 200, 500 μg or 1 mg), and 14 days later, spleen andlymph nodes were removed and the cells assayed for proliferation andcytokine secretion after 4 days in culture with BiP.

[0128] Results: It was evident from these experiments that the optimalimmunising dose of BiP was 500 μg as T cells derived from micere-stimulated in vitro with 10 μg/ml BiP proliferated most vigorously(600,000 CPM) whereas T cell proliferation from all other immunisationgroups were below 40,000 CPM. Cytokines studies were in agreementshowing levels of IL-4 reaching ˜800 pg/ml in mice immunised with 500 μg(below 500 pg/ml in all other groups). Similarly IL-5 levels reached˜600 pg/ml in the 500 μg group and did not reach over 400 pg/ml in anyof the other groups. T cells from naïve mice proliferated very slightlyto BiP stimulation, produced very low quantities of Th2 cytokines and˜1000 pg/ml of IFN-γ.

[0129] Conclusion: The optimum proliferation and cytokine secretion uponin vitro BiP stimulation was observed when mice were immunised with 500μg of BiP.

1. The use of BiP or a functional fragment or homolog thereof, or anucleic acid molecule encoding BiP or a functional fragment or homologthereof, in the manufacture of a medicament for the treatment orprevention of an unwanted immune response.
 2. The use according to claim1, wherein the unwanted immune response is associated with animmune-mediated disease.
 3. The use according to claim 2, wherein theimmune-mediated disease is an auto-immune disease.
 4. The use accordingto claim 2, wherein the immune-mediated disease is type 1 diabetes,thyroiditis or multiple sclerosis, systemic lupus erythematosus, Crohn'sdisease and all forms of viral and autoimmune hepatitis.
 5. The useaccording to any one of claims 2 to 4, wherein the medicamentadditionally comprises an agent for enhancing the treatment orprevention of the immune-mediated disease.
 6. The use according to anyone of claims 2 to 5, wherein the medicament is administered to anindividual suffering from or susceptible to developing animmune-mediated disease.
 7. The use according to claim 6, wherein themedicament is administered to the individual prior to the development ofthe immune-mediated disease or as soon as the immune-mediated diseasehas be diagnosed.
 8. The use according to claim 1, wherein the unwantedimmune response is associated the rejection of a transplanted organ, atissue or cells.
 9. The use according to claim 8, wherein the medicamentadditionally comprises an agent for enhancing the treatment orprevention of transplant rejection.
 10. The use according to claim 9,wherein the agent is a suppressor of the immune system.
 11. The useaccording to any one of claims 8 to 10, wherein the medicament isadministered to an individual before or at substantially the same timeas the transplantation of an organ, tissue or cells.
 12. The useaccording to claim 1, wherein the unwanted immune response is the immuneresponse to a biologic.
 13. The use according to claim 12, wherein thebiologic is a therapeutic protein, glycoprotein or carbohydrate.
 14. Theuse according to claim 12 or claim 13, wherein the medicamentadditionally comprises an agent for enhancing the treatment orprevention of the immune response to the biologic.
 15. The use accordingto any one of claims 12 to 14, wherein the medicament is administered toan individual before or at substantially the same time as the biologic.16. The use of BiP or a functional fragment or homolog thereof, or anucleic acid molecule encoding BiP or a functional fragment or homologthereof, for stimulating the release of IL-0 from cells capable ofreleasing IL-10.
 17. The use according to claim 16, wherein BiP or afunctional fragment or homolog thereof, or a nucleic acid moleculeencoding BiP or a functional fragment or homolog thereof, additionallystimulates gene expression of at least one of monocyte migrationinhibitory factor (MIP), soluble TNF receptor II, IL-10anti-inflammatory mediators and tissue inhibitor of matrixmetalloproteinases (TIMP).
 18. The use according to claim 17, whereinBiP or a functional fragment or homolog thereof, or a nucleic acidmolecule encoding BiP or a functional fragment or homolog thereof, doesnot stimulate gene expression of matrix metalloproteinases (MMPs),monocyte chemoattractant protein (MCP-1) or TNFα.
 19. A pharmaceuticalpreparation comprising BiP or a functional fragment or homolog thereof,or a nucleic acid molecule encoding BiP or a functional fragment orhomolog thereof, in combination with a pharmaceutically acceptablecarrier for use in the treatment or prevention of an unwanted immuneresponse.
 20. The pharmaceutical preparation according to claim 19,wherein the unwanted immune response is associated with animmune-mediated disease; associated with the rejection of a transplantedorgan, tissue or cell; or is the immune response to a biologic.
 21. Thepharmaceutical composition according to claim 19 or claim 20, whichadditionally comprises an agent for enhancing the treatment ofprevention of the unwanted immune response.
 22. A method of treating orpreventing an unwanted immune response comprising administrating to anindividual in need of such treatment an effective dose of thepharmaceutical composition according to any one of claims 19 to
 21. 23.The use of BiP or a peptide fragment thereof (including a syntheticpeptide) for stimulating the release of Interleukin 10 (IL-10).
 24. Theuse according to claim 23, for the preparation of a medicament for thetreatment of auto-immune disease.
 25. The use according to claim 24, forthe preparation of a medicament for the prevention of rejection oftransplanted organs, tissues, or cell.
 26. The use according to claim23, for the preparation of a medicament for the prevention of an immuneresponse to biologic therapeutic substances.
 27. The use according toclaim 23, for the preparation of a medicament for the treatment ofimmune-mediated disease, including type 1 diabetes, thyroiditis, andmultiple sclerosis, systemic lupus erythematosus, Crohn's disease andall forms of viral and autoimmune hepatitis.
 28. Pharmaceuticalpreparations comprising BiP or a fragment thereof adapted for treatmentof the diseases or situations referred to in any of claims 23 to
 27. 29.A method of treatment of the diseases or situations referred to in anyof claims 23 to 27, which comprises administering to a patient in needthereof. BiP or a fragment thereof.